Ribonuclease H (RNase H) is an essential activity for the lifecycle of retroviruses such as HIV, and therefore represents an important drug target. The goal of this proposal is to determine the relationship between structure and function in the RNase H domain of HIV-1 reverse transcriptase. The research approaches include a variety of biophysical and genetic experiments. The isolated RNase H domain of HIV reverse transcriptase is known to fold into a RNase H-like structural motif, yet the refolded enzyme is inactive. Instability in the C-terminus and a missing basic protrusion have been postulated by the PI to play an important role in this lack of activity. In order to obtain selective inhibitors of HIV RNase, an active isolated domain is necessary. Active variants of this domain will be generated by a combination of rational design and genetic selections. One such Mn++-dependent domain has already been obtained by the applicant in preliminary results. The structure and stability of these active variants will be characterized by circular dichroism, fluorescence, NMR and crystallographic structural studies. The special role of the C- terminal region will be investigated further. Preliminary studies reveal that peptides from this region of HIV are unstructured, whereas similar peptides from the active E. coli homologue and MoLV are well folded. Effects of mutations on both the isolated domain and the peptides will be characterized. Peptide inhibitors will be designed that compete with this region of the protein. In an attempt to characterize and interfere with the structure/function relationship, the equilibrium and kinetic folding properties of the isolated RNase H domain will also be investigated. Since the asymmetric post-translational processing of the HIV reverse transcriptase molecule appears to require one of the RNase H domains to be at least partially unfolded, these studies may also shed light on this mechanism.The ultimate aim of the proposed research is selective inhibition of this essential retroviral activity.